Troubleshooting

Transfection Problem

Possible Cause

Suggested Solution

Low transfection efficiency.

Not using optimal cationic lipid reagent.

Select the cationic lipid reagent likely to result in the highest transfection. efficiency for your cell type. See the references listed in the “Transfection Collection” on the web at www.invitrogen.com find the best reagent for your cell type.

Low transfection efficiency.

Not using optimal cationic lipid reagent concentration, DNA concentration, time or cell density.

Optimize these parameters under final transfection conditions. See the cell-specific transfection protocols on the web at www.invitrogen.com.

Low transfection efficiency.

DNA-cationic lipid reagent complexes not formed.

Do not use serum during complex formation step. Opti-MEM® I Medium or D-MEM are good media for complex formation. If using serum containing medium for transfections, form the complex in the absence of serum. Note: Do not use Opti-MEM® I Medium with Lipofectamine® with Plus™ Reagent or for insect cells. Sf-900 II SFM gives optimal results for Sf9 or Sf21 cells. For D. mel-S2 cells, use Drosophila-SFM.

Low transfection efficiency.

Inhibitors were present.

Do not use antibiotics, EDTA, citrate, phosphate, RPMI medium, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare complexes.

Low transfection efficiency.

Cationic lipid was frozen.

Do not use cationic lipid reagents that have been frozen. Store at +4°C.

Low transfection efficiency.

Improper cell density.

Cell density should be 70% to 90% confluent at time of transfection. (30%-70% confluent if using Optifect™ Transfection Reagent)

Low transfection efficiency.

Problems with the transfection assay.

Include a positive control for the transfection assay.

Low transfection efficiency.

The promoter-enhancer of the transfected DNA is not recognized by the cell type.

Make sure the transfected DNA is compatible with the target cell type.

Low transfection efficiency.

For Lipofectin® Reagent, no preincubation of cationic lipid with medium.

Diluted Lipofectamine® 2000 Reagent was incubated too long before mixing with diluted DNA.

For dilutions prepared in Opti-MEM® I Medium, be sure that the diluted Lipofectamine® 2000 Reagent is combined with the diluted DNA within 30 min. If D-MEM is used as the diluent, mix with the diluted DNA within 5 min.

Low transfection efficiency.

Cell density too low for Lipofectamine® 2000 Reagent transfection.

Use cells at 90% confluency for Lipofectamine® 2000 Reagent transfections.

High cell death (toxicity)

Too much DNA.

Perform a dose-response curve to determine the optimal amount of DNA. Include a cationic lipid reagent in the dose-response transfections, as DNA alone has a minimal effect on cell growth.

High cell death (toxicity)

Too much cationic lipid reagent.

Perform a dose-response curve to determine the optimal amount of cationic lipid reagent. Include DNA with the cationic lipid reagent in the dose-response experiment, as cationic lipid reagent alone has a minimal effect on cell growth.

High cell death (toxicity)

Too few cells.

Perform a dose-response curve to determine the optimal number of cells per transfection. Balance cell number with efficiency for your application.

High cell death (toxicity)

Cell viability decreased without serum.

Use Opti-MEM® I medium. Reduce or omit the number of washes in serum-free medium. Use 5 to 10% serum in the transfection medium. Be sure to form complexes in the absence of serum.